Global genome initiatives including the Human Genome Project have generated enormous amounts of information, spawned new technologies and catalyzed the emergence of a new discipline in biology. Systems biology attempts to build biological knowledge from the global analysis of biological systems and pathways from genes to proteins. To fuel this growing discipline, there is a need to develop means of global assessment that will ultimately clarify our understanding of protein function. Because both protein activity and turnover are closely tied to protein post-translational modification (e.g., phosphorylation and ubiquitination), new technologies are required for the large-scale identification, characterization, and quantification of protein post- translational modifications. Reversible protein phosphorylation is a general process affecting most cellular regulatory systems. Phosphorylation is critical to maintaining normal physiology;malfunctions in protein phosphorylation have been implicated in the etiology of many diseases as diverse as diabetes, cancer, and Alzheimer's disease. Protein phosphorylation is an intense research area with great promise for improving human health. Technologies developed under the previous cycle of this grant allowed for the rapid, sensitive and accurate identification of thousands of phosphorylation sites from a single sample. This landmark achievement brings other important unsolved issues to light. Two of the most critical are addressed in this proposal - quantitative phosphorylation profiling and phosphorylation site occupancy. For Aim 1, we will develop quantitative strategies to study dynamic phosphorylation on a near-global scale. This includes combining and integrating a robust stable isotope labeling strategy, new enrichment approaches, and new software tools with our existing platform. For Aim 2, we will perform a highly focused set of experiments to globally profile phosphorylation differences after DNA damage in both yeast cells and human cell lines. These experiments will provide a biologically important proving ground for newly developed techniques and software. Finally, for Aim 3, we will develop and apply a strategy to determine the basal site occupancy levels for all kinases (87) in the tandem affinity purification (TAP) yeast library. In addition, site occupancy levels for a set of human kinases will be examined for DNA damage- and cell cycle-dependency. PUBLIC HEALTH RELEVANCE Protein phosphorylation is an intense research area with great promise for the improvement of human health. In addition to its critical role in normal physiology, malfunctions in protein phosphorylation have been implicated as a contributing factor in the causation of many diseases as diverse as diabetes, cancer, and Alzheimer's disease. This proposal will provide new technologies to profile phosphorylation events between different cell states on a global scale.